Trypanosoma brucei is a group of pathogenic protozoans causing a spectrum of infectious human and related animal diseases that can be fatal and economically devastating. The long-term goal is to elucidate the biological importance and biochemical function of the editosomal proteins KREPA6 and KREPA5 in mitochondrial RNA editing, a unique RNA processing mechanism in trypanosomes that is essential for their survival. The proposal's specific aims are (1) to determine the physiological effects of disrupting the expression of these genes, (2) assess their RNA binding properties, and (3) identify KREPA6 protein binding partners. Regulatable RNAi, gene knock-outs, and construction of mutant transgenic strains will be used to determine their effects on cell growth and survival, editosome integrity and RNA editing in vivo. Recombinant proteins will be used to characterize their RNA binding substrates and protein binding partners in vitro. These studies will help reveal how KREPA5 and KREPA6 might contribute to the coordination of RNA substrate binding activities and catalytic steps within the editosome during RNA editing, helping advance our mechanistic understanding of RNA editing in trypanosomes.